Treatment of hi-virus infections with oxidised blood proteins

ABSTRACT

The invention relates to the field of medicaments for combating an infection of a host cell by HI viruses and/or for inhibiting binding of an Env protein to a CD4 protein. For these purposes, the invention provides medicaments which comprise oxidized proteins, oxidized peptides and/or peptidomimetics of such oxidized proteins and/or oxidized peptides, as well as preparation processes for such medicaments and therapeutic and non-therapeutic possible uses of these medicaments.

CROSS REFERENCE TO RELATED APPLICATION

This application is a national stage of PCT/EP2003/004374 filed Apr. 25,2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of medicaments for combating aninfection of a host cell by HI viruses and/or for inhibiting binding ofan Env protein to a CD4 protein. For these purposes, the inventionprovides medicaments which comprise oxidized proteins, oxidized peptidesand/or peptidomimetics of such oxidized proteins and/or oxidizedpeptides, as well as preparation processes for such medicaments andtherapeutic and non-therapeutic possible uses of these medicaments.Oxidized proteins, oxidized peptides and the abovementionedpeptidomimetics are called collectively by the term “oxP” in thefollowing.

2. Related Art of the Invention

As described in WO 02/22150 A2 and WO 02/32445 A2, oxP, such as, but notlimited to, “immune defence activated” antithrombin (IDA-ATIII),oxidized serum albumin, oxidized fibrinogen, bind to the GP120 of theEnv protein in the envelope of the HI virus. As demonstrated by us in WO02/22150 A2 by way of example for IDA-ATIII, oxPs are capable ofpreventing the multiplication of the HI virus in the host cell.

SUMMARY OF THE INVENTION

In the context of the present invention, we demonstrate that oxPs arecapable of preventing contact between the HI virus and the host cell, ofblocking the formation of syncytia from infected and non-infecteddefence cells and therefore of already inhibiting the infection at the“entry” level. Surprisingly, it has furthermore been found that nopurified oxidized proteins and oxidized peptides, such as human serumalbumin, are necessary to suppress infection of a host cell with HIviruses, but that oxidized blood plasma as such can already prevent aninfection at the “entry” level.

Although some potent antiviral medicaments exist, HIV has developed intoa true worldwide pandemic and has therefore become the most importantinfectious disease. Over 4 million people currently die from thisdisease every year. Since HIV medicaments which are approved today, suchas protease inhibitors and reverse transcriptase inhibitors, are notcapable of removing HIV completely from infected persons, there is theurgent need to discover novel antiviral medicaments.

HIV infections/AIDS represent one of the most important crises in thedevelopment of humanity. An HIV medicament must therefore be foundwhich, in addition to meeting all the scientific requirements, on theone hand is easy to prepare and is attainable, and on the other hand canbe made readily accessible to people specifically in the sub-Sahararegions.

These problems have been solved with the present invention, since theoxPs provided here already block HIV infections at the very first entrylevel, oxPs are easy and cheap to prepare (protein from the plasma of ahuman can be used directly) and this process can be carried out not onlyin special laboratories.

Enveloped viruses in principle penetrate into their target cell in thatthe membrane of the virus comes into contact with the membrane of thetarget cell and the two membranes then become so close to one anotherthat they fuse. For contact of the HIV-1 virus with the host cell,binding of the external HIV envelope protein Env to the CD4 receptor isnecessary. This takes place via the Env constituent GP120. GP120subsequently binds to one of the main co-receptors CXCR4 or CCR5. Thebinding of GP120 to its receptors leads to a change in the conformationof the external GP120/GP41 complex. This interaction is a basicprerequisite for rendering possible the provision of the viral GP41 Nterminus, which in the end leads to the membrane fusion.

Neutralizing antibodies which are directed directly against GP120 areone factor which can partly block the viral entry. However, neutralizingantibodies of natural infections have only a limited efficiency, sinceon the one hand HIV produces a very large number of different antigenvariants, and on the other hand after an infection a restricting “clonaldominance” of HIV-neutralizing antibodies occurs. New HIV-1 variants cantherefore easily escape such a highly specific but restricted response.

The most recent findings have shown that the non-specific inheritedimmune response is relevant for defence against HIV. Polymorphonuclearneutrophilic leukocytes (PMNL) and monocytes are viricidal towards HIV-1after stimulation. Lipopolysaccharide (LPS) stimulation, which leads tooxidative bursting of leukocytes, causes a blockade of the HIV entry,without taking into consideration the viral co-receptor phenotype.

Leukocytes generate H₂O₂ and secrete the haem protein myeloperoxidase(MPO). Klebanoff and his colleagues have shown that stimulated PMNL frompatients with inherited deficiency of the enzyme MPO had a reducedviricidal activity. By addition of MPO, it was possible to reconstitutethe reduced defence power again. On the basis of the current state ofknowledge, it is assumed that the MPO product HOCl itself is the agenthaving an antiviral action. The expression and the release of MPO, whichproduces HOCl, is strictly controlled in vivo. Free HOCl represents apart of oxidative stress.

In the context of the invention, we have now found that protein/peptideswhich has/have come into contact with HOCl can be transformed into anantiviral form, and as a result HOCl has an indirect action againstHIV-1, in addition to the known direct action.

Relevance of this type of inhibition for future therapeuticapplications: HOCl, which is produced by MPO in inflamed tissue,modifies LDL and many other human proteins in vivo. HOCl-modifiedproteins may be detected by specific monoclonal antibodies (WO-02/32445A2). A structural change generated by HOCl treatment therefore providesan epitope which is already present in vivo and therefore well-known tothe immune system. OxPs should therefore be tolerated in vivo.

In addition, oxP is based on a relatively simple chemical modificationassociated with low costs.

Why does the body not produce enough oxP in vivo to protect itself fromHIV infection? One answer to this question could be the observation thatthe function of the neutrophilic leukocytes and of the monocytesdeteriorates in HIV-infected persons at the start of the infection, andthat the losses of function correlate positively with theextent/progression of the HIV-induced disease.

The object of the present invention was therefore to provide amedicament which already inhibits an HIV infection at the “entry” level,and at the same time is easy and inexpensive to prepare and thereforecan also be used for patients in the so-called “third world”.

The object is achieved by a preparation process for a medicament forcombating an infection of a host cell by HI viruses and/or forinhibiting binding of an Env protein to a CD4 protein, which ischaracterized by the steps:

-   -   a) provision of a mixture, comprising proteins and/or peptides,        which can be obtained from whole human and/or animal blood    -   b) oxidation of the proteins and/or peptides contained in the        mixture.

While in earlier studies the action of individual, purified proteins onthe binding of an Env protein to a CD4 protein was demonstrated, it hasnow emerged, surprisingly, that the infection of a host cell by HIviruses and/or the binding of an Env protein to a CD4 protein can alsobe inhibited, reduced or even completely suppressed by protein mixtures.In particular, these actions can also be achieved by complex protein-and/or peptide-containing mixtures having more than one variety ofprotein or peptide. This was surprising, since it was to be expectedthat the process for the preparation of the oxidized proteins and/orpeptides necessary for combating an infection of a host cell by HIviruses and/or for inhibiting the binding of an Env protein to a CD4protein is impaired during the oxidation in such complex mixtures byachieving false conformations and by side reactions such that theactivity of oxidized proteins in such mixtures is reduced or eliminatedcompletely.

In the context of this invention, whole blood is the blood taken from ahuman or animal, in particular a monkey, cow, sheep, pig, dog, goat,rabbit or mouse, and optionally mixed with a suitable anticoagulant.

In the context of the present invention, as already stated above, it hasbeen found that oxP blocks the binding of HIV-GP120 to CD4 and thusprevents the penetration of the HIV into the target cell. Since a virusis dependent upon penetration into a host cell for its multiplication,however, it cannot multiply further. The formation of syncytia fromHIV-infected defence cells with non-infected defence cells does not takeplace, so that these pursue their task further and can destroy infectedcells.

It has proved to be particularly advantageous if the mixture oxidized instep b) comprises a plasma protein and/or a plasma peptide which is orare oxidized in step b). Plasma proteins and plasma peptides here arethose proteins and peptides which are contained in the liquid whichsettles on the top on centrifugation or sedimentation of whole blood,and the proteins, peptides and protein or peptide complexes which can beprepared from them. The plasma proteins and plasma peptides include, inparticular, serum albumin, in particular human serum albumin and bovineserum albumin, antithrombin, “immune defence activated” antithrombin(IDA-ATIII), fibrinogen, coagulation factors and immunoglobulins. It istherefore furthermore preferable to use a plasma protein, plasmapeptide, serum albumin, in particular human serum albumin and bovineserum albumin, antithrombin, “immune defence activated” antithrombin(IDA-ATIII), fibrinogen, a coagulation factor or an immunoglobulin ormixtures of these substances for the preparation of a medicament forcombating an infection of a host cell by HI viruses and/or forinhibiting binding of an Env protein to a CD4 protein.

Instead of or in addition to oxidized proteins and/or oxidized peptides,the medicament can also be prepared by mixing of a pharmaceuticallyacceptable carrier with a peptidomimetic, which can replace an oxidizedprotein and/or an oxidized peptide of a mixture oxidized in step b) byone of the processes according to the invention which are describedabove. Research into pharmaceutical chemistry, in particular, hasproposed strategies for the preparation of peptidomimetics (cf. Rompponline, document identification RD-16-00950). By using these strategies,it is possible for the person skilled in the art to produce thepeptidomimetics necessary for carrying out the preparation processaccording to the invention.

The medicament prepared by the process thus comprises oxidizedprotein(s), oxidized peptide(s), oxidized amino acids, peptidomimeticsand/or peptide analogues (summarized as oxP).

In the context of the present invention, the medicament can compriseeither the complete oxP, which can be prepared, for example, by theprocess described in the examples. However, oxP formed after defencereactions can also be isolated from the body. Furthermore, plasmafractions (plasma protein mixtures) of the patient himself or of adonor, without necessary isolation of the proteins, can be converteddirectly into a medicament which comprises oxP. It is furthermoreconceivable to use ox-peptides which bind to HIV-GP120. Analogues of oxPare also suitable in the context of the present invention if theylikewise prevent the entry of HIV into its target cells.

A medicament according to the invention can of course comprise furtherpharmaceutically acceptable auxiliary or/and carrier substances, themedicament being formulated for local, intradermal, superficial,intraperitonal, intravenous, intramuscular or oral administration orrendering possible its administration via vesicles. The medicamentaccording to the invention therefore preferably comprises thoseauxiliary and carrier substances which render possible the particularpreferred mode of administration.

The medicament according to the invention can of course comprise, inaddition to oxP, parts or analogues or mimetics thereof, furthersubstances, such as, for example, antibiotics, other HIV infectioninhibitors, etc. Depending on the concomitant disease to be treated, itmay be of advantage to provide supporting treatment with knownmedicaments. An appropriate combination of this medicament with oxP istherefore optionally a preferred embodiment of the present invention.

Advantageous uses of the medicaments according to the invention aredescribed in the claims. A particularly advantageous non-therapeutic usecomprises the treatment of cells and/or cell cultures, in each case inparticular of animal or human origin, for combating an infection of ahost cell by HI viruses, in particular by inhibition of the binding ofan Env protein to a CD4 protein, and in this context in particular forinhibiting the binding between a GP120 unit of an Env protein to a CD4protein. The medicaments according to the invention can furthermore beused for binding and optionally for detection of an Env protein, inparticular a GP120 unit of an Env protein. The invention is described inmore detail in the following with the aid of the examples and thefigures.

1.) Example for the preparation of oxP (see also our Application WO02/32445 A2)

In order to transform normal human serum albumin into the antiviralform, HSA was activated with HOCl. Freshly prepared HOCl was added toHSA in a molar ratio of 1:100. After an incubation time of 30 minutes atroom temperature, the hypochlorite which remained was removed by gelfiltration (Sephadex G25).

In another use, protein mixtures were isolated from human plasma by astandard method (e.g. ammonium sulfate precipitation/desalination orcryoprecipitation) and these were then modified directly with freshlyprepared HOCl, as described for human serum albumin.

2.) OxP is not cytotoxic.

The HOCl-modified HSA was first tested in the ³H-tymidine incorporationassay. Up to a use concentration of 50 μg/ml, no anticellular activityin respect of the cell proliferation of Hela or GHOST cells comparedwith normal HSA was to be observed (FIG. 1).

3.) The binding of oxP to IIIB GP120 (from the AIDS reagent EVA project)was illustrated in a standard ELISA assay (FIG. 2 a). In addition, thebinding of oxP both to IIIB GP120 and to SF2 GP120 was demonstrated insurface plasmon resonance spectroscopy (SPR) (FIG. 2 b). In bothexperiments, the direct interaction of oxP on GP120 was demonstrated.Only after transformation of the protein into the oxP form was aspecific binding to be observed. If normal protein, in this case HSA,normal bovine serum albumin, glutathione S-transferase (GST) or fusionprotein with a GST-VS, which included the FP120 V3 loop, was used, nobinding was to be observed. In all these control experiments, the“response units” (RU) were <5. In addition to the SPR binding study, thekinetics of the oxP (here ox ATIII) GP120-IIIB interaction wereinvestigated. The analysis gave ka and kd values of 1.47*10⁻⁹ and7.01*10⁻¹⁰ M, and resulting from these a KD of 7.0*10⁻¹⁰ M (Rmax=120;Chi2=40).

4.) A non-fractionated protein mixture from human plasma binds toHIV-GP120 after treatment with HOCl, as described above. (FIG. 3)

5.) OxP neutralizes HIV

For HIV neutralization experiments, the HIV-1 strains NL4-3 and avariant of the NL4-3, NL-991, in which the V3 loop was exchanged for aV3 loop of the primary isolate PI-991, were used. NL4-3 is a monotropicvirus which uses only the CXCR4 co-receptor. The NL-991 virus isR5-monotropic and uses only CCR5 as a co-receptor. FIG. 4 shows that thereplication of both viruses is inhibited by oxP. This is reflected inthe amount of HIV p24 antigen produced.

6.) In HIV cell cultures, HIV-infected cells fuse with non-infected CD4⁺target cells. This fusion among cells is known as syncytia formation.This syncytia formation is to be attributed to the binding of GP120,which is expressed on the membrane of infected cells, to the CD4receptor on the target cell and subsequent insertion of the GP41 Nterminus into the target membrane. Both viruses which were used in thisneutralization assay (NL4-3 and NL-991) were capable of forming syncytiawith the GHOST-CXCR4 and GHOST-CCR5 cells (FIG. 5). It was possible forboth the syncytia formation induced by the NL4-3 virus (FIG. 5 a), andthat induced by the NL-991 virus (FIG. 5 g), to be inhibited by additionof oxP (in this case ox-HSA) in concentrations of up to 20 μg/ml (FIG. 5e-4 k). oxP showed a dose-dependent inhibition (5 b-e; 5 h-k). Asalready shown in FIG. 1, oxP itself influenced neither cellproliferation nor cell morphology (FIG. 5 f; 5I), and the staining ofthe cell nuclei proved the vitality of the GHOST cells.

7.) OxP blocks HIV infection at the “entry level”.

Syncytia formation is based on the presence of the viral envelope andthe viral docking proteins on the membrane surface of the host cells.Hela-P4 cells (CD4+, CXCR4+, CCR5+), which additionally expressed theviral receptors GP120/GP41, were therefore used. For this, Hela-P4 cellswere transfected with GP160 vectors, so that they expressed the Envproteins of HIV-NL4-3 and HIV-NL-911. Gp160-transfected Hela-P4 cellsfused and, after 28 h, in contrast to non-transfected cells (6 b; 6 h),formed syncytia (FIG. 6 a; 6 g).

Incubation with oxP led to a dose-dependent inhibition of the formationof syncytia (6 d, f, 6 j, l), in contrast to incubation withnon-modified protein (in this case, as an example, HSA) (6 c, e, i, k).This transfection assay imitates the entry of X4- and R5-tropic HIviruses (NL4-3, NL-911). In both syncytia test methods, oxP showed“anti-HIV entry” activity at 20 and 50 μg/ml. This illustrates that oxPacts at the GP120-CD4 interaction level with an ID>95 at 50 μg/ml.

8.) Precipitation of plasma proteins and oxidation thereof:

10 ml citrate blood were centrifuged at 3,200 rpm/2,000 g for 10 minutesand the supernatant plasma was removed. 5 M (NH4)2SO4 was added in avolume ratio of 1:1, the plasma was stirred for 20 minutes and theplasma proteins were thereby precipitated. The suspension wascentrifuged again for 10 minutes at 3,200 rpm/2,000 g, the supernatantwas discarded and the precipitated proteins were resuspended in PBSbuffer (pH 7.4). The solution was introduced into a dialysis hose(exclusion limit 10,000 D) and dialysed against PBS buffer for 3 days.During this procedure, the buffer was changed 3 times. Alternatively,the protein mixture was desalinated by a gel filtration process.

After the dialysis/gel filtration, the total protein content wasdetermined photometrically by standard methods.

Oxidation to the medicament according to the invention: Fresh HOCl (12μl) was added to 500 μg of the protein solution and the mixture wastopped up to a volume of 1 ml with PBS/0.1 mM EDTA buffer. After areaction time of 15 minutes on ice (0° C.), the solution was introducedonto a gel filtation column equilibrated with PBS buffer and theunreacted HOCl was removed in this way.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1: IDA-HSA is not cytotoxic

Hela cells which express human CD4, CXCR4 and CCR5 were cultured in thepresence of [³H]-tymidine and various concentrations of IDA-HSA (□) orHSA (▴). The cells were sown at 10⁴ cells per “well” in a 96 “well”plate in triplicate. On day 2, [³H]-tymidine (10 μCi/ml) was added toeach “well”. After 8 hours, the DNA was harvested and bound to a glassfibre membrane and the [³H]-tymidine incorporated was quantified with aβ-counter.

FIG. 2: IDA-HSA binds to HIV-1 GP120

The specific binding of IDA-HSA to GP120 was illustrated in a standardELISA method (a) and by surface plasmon resonance spectroscopy (SPR)(b). For the ELISA, a 96-well plate was coated with 100 μl/well ofrecombinant GP120 (1 μg/ml) and then blocked with 0.25% gelatine (inPBS) (1 h RT). IDA-HSA (◯) and HSA (●) were applied in variousconcentrations (0, 0.25, 0.5, 1, 2, 5, 10, 20 μg/ml in PBS). Boundprotein was detected with HRP-conjugated, polyclonal, specific anti-HSAantibodies (Sigma). For the SPR, GP120 (10 μg/ml) was bonded covalentlywith EDC/NHC on a dextran-coated, CH-activated sensor chip (CM5,Biacore, Sweden). The flow rate was 5 μl/min for 10 min. After blockingwith ethanolamine, the binding of IDA-HSA and HSA (in each case 1 μg/ml)was analysed at a flow rate of 5 μl/min for 6 minutes.

FIG. 3: Oxidized plasma protein mixtures bind to HIV-GP120

The specific binding of a mixture of oxidized plasma proteins to GP120was demonstrated by surface plasmon resonance spectroscopy (SPR). GP120(10 μg/ml) was bonded covalently on a sensor chip (C1, Biacore, Sweden).Injection of 20 μl 100 mM glycine, Purification of the sensor 0.3%Triton X-100 pH 12 (twice) surface 5 μl/min Flow rate Injection of 20 μl400 mM EDTA Removal of calcium ions from the sensor surface Injection of50 μl NHS/EDC Activation of the sensor surface Injection of 20 μl 100 nMP120 Covalent coupling of P120 in 10 mM NaAc pH 4 (dilution 1:10)Injection of 55 μl ethanolamine Blocking of the remaining activatedesters

A solution of 93.5 nM PluO was then injected via immobilized P120. Anunambiguous binding curve (see above) was obtained. In subsequentexperiments, it was shown that the resulting signal height correlateswith the amount of immobilized P120.

FIG. 4: Inhibition of the HIV replication

GHOST-CXCR4 or GHOST-CCR5 cells were infected with X4-tropic NL4-3(triangles) or R5-tropic NL-991 viruses (squares) (500TCID₅₀). On day 5,the cell culture supernatant was tested for p24 antigen with a p24standard ELISA. The mean of 3 measurements is shown. The standard errorfor the mean was <10%, NL4-3+IDA-HSA (A); NL4-3+HSA (Δ); NL-991+IDA-HSA(▪); NL-991+HSA (□).

FIG. 5: Inhibition of the HIV-induced syncytia formation GHOST-CXCR4 orGHOST-CCR5 cells were infected with 500 TCID₅₀ of the HIV laboratoryisolates (A-E) NL4-3 (X4-monotropic) or (F-L) NL-991 (R5-monotropic).The infection was inhibited by addition of IDA-HSA protein to theculture medium with a final concentration of 0, 2, 5, 10 or 20 μg/ml. 5days after the start of infection, the infection was rendered visible bydemonstration of the syncytia induced and of the destruction of the celllawn. For this, the cells/nuclei were stained by a standardeosin/methylene blue/azure staining procedure (Hemacolor, Merck). (a),NL4-3 infected cells; (b) NL4-3+2 μg/ml IDA-HSA; (c) NL4-3+5 μg/mlIDA-HSA; (d) NL4-3+10 μg/ml IDA-HSA; (e) NL4-3+20 μg/ml IDA-HSA; (g)NL-991 infected cells; (h) NL-991+2 μg/ml IDA-HSA; (i) NL-911+5 μg/mlIDA-HSA; (j) NL-911+10 μg/ml IDA-HSA; (k) NL-911+20 μg/ml IDA-HSA; (1)20 μg/ml IDA-HSA.

FIG. 6: Inhibition of the GP160-induced syncytia formation

Hela cells which express human CD4, CXCR4 and CCR5 were transfected withpSVATGrev plasmids, which express NL4-3 or NL-911 env. For this, eitherIDA-HSA or HSA protein was added (final concentration 20 and 50 μg/ml).The syncytia formation was investigated after 28 hours by standard phasecontrast microscopy of the living cells. IDA-HSA prevented syncytiaformation in a dose-dependent manner.

REFERENCES

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1-9. (canceled)
 10. A medicament for combating an infection of a host cell by HI viruses and/or for inhibiting binding of an Env protein to a CD4 protein, said medicament prepared by the steps: a) providing a mixture comprising human and/or animal blood plasma, and b) oxidating the proteins and/or peptides of the blood p plasma which are contained in the mixture with HOCl.
 11. A process for the preparation of a medicament for combating an infection of a host cell by HI viruses and/or for inhibiting binding of an Env protein to a CD4 protein, said process comprising: a) providing a mixture comprising human and/or animal blood plasma and b) oxidizing the proteins and/or peptides of the blood plasma which are contained in the mixture with HOCl.
 12. A process according to claim 11, wherein one of the proteins contained in the mixture and oxidized in step b) is serum albumin.
 13. A process as in claim 11, wherein said serum album is human serum albumin or bovine serum albumin, antithrombin, immune defence activated antithrombin (IDA-ATIII), fibrinogen, coagulation factor or an immunoglobulin.
 14. A process as in claim 11, said process including mixing of a pharmaceutically acceptable carrier with a peptidomimetic which can replace an oxidized protein and/or an oxidized peptide of a mixture oxidized in step b) according to one of the preceding claims.
 15. A process as in claim 11, further comprising mixing the obtained mixture with a peptidomimetic which can replace an oxidized protein and/or an oxidized peptide of a mixture oxidized in step b).
 16. A method for non-therapeutic combating of an infection of a host cell by HI viruses, comprising administering to a subject in need thereof a medicament prepared by the steps: a) providing a mixture comprising human and/or animal blood plasma, and b) oxidating the proteins and/or peptides of the blood p plasma which are contained in the mixture with HOCl.
 17. A method for for non-therapeutic inhibition of binding of an Env protein to a CD4 protein, comprising administering to a subject in need thereof a medicament prepared by the steps: a) providing a mixture comprising human and/or animal blood plasma, and b) oxidating the proteins and/or peptides of the blood p plasma which are contained in the mixture with HOCl.
 18. A method for non-therapeutic combating of an infection of a host cell by HI viruses, comprising administering to a subject in need thereof a medicament prepared by the steps: a) providing a mixture comprising human and/or animal blood plasma, and b) oxidating the proteins and/or peptides of the blood p plasma which are contained in the mixture with HOCl.
 19. A method as in claim 15, wherein said method is for non-therapeutic inhibition of binding between a GP120 unit of an Env protein to a CD4 protein. 